The innate-immune restriction factor MxA inhibits influenza replication by targeting the viral nucleoprotein (NP). Human influenza virus is more resistant than avian influenza virus to inhibition by human MxA, and prior work has compared human and avian viral strains to identify amino-acid differences in NP that affect sensitivity to MxA. However, this strategy is limited to identifying sites in NP where mutations that affect MxA sensitivity have fixed during the small number of documented zoonotic transmissions of influenza to humans. Here we use an unbiased deep mutational scanning approach to quantify how all single amino-acid mutations to NP affect MxA sensitivity in the context of replication-competent virus. We both identify new sites in NP where mutations affect MxA resistance and re-identify mutations known to have increased MxA resistance during historical adaptations of influenza to humans. Most of the sites where mutations have the greatest effect are almost completely conserved across all influenza A viruses, and the amino acids at these sites confer relatively high resistance to MxA. These sites cluster in regions of NP that appear to be important for its recognition by MxA. Overall, our work systematically identifies the sites in influenza nucleoprotein where mutations affect sensitivity to MxA. We also demonstrate a powerful new strategy for identifying regions of viral proteins that affect inhibition by host factors.

ppat.1006288.g006: A mutation at site 51 in NP increases MxA sensitivity as measured by viral competition.We used reverse genetics to generate viruses carrying the wildtype NP, the D51N mutation, or a synonymous mutation at site 51. Each virus was generated in duplicate. We mixed the wildtype virus with one of the mutant viruses, infected MxA-expressing and non-expressing cells, and measured mutant frequencies after 10 and 54 hours. The plots show the frequency of the wildtype D51 variant relative to the N51 amino-acid mutant or the Dsyn51 synonymous mutant at each timepoint. In both replicates of the competition, the wildtype D51 variant was strongly favored over the N51 mutant in MxA-expressing cells. We targeted a 1:1 ratio of infectious particles in our initial inoculum, but this ratio was impossible to verify by direct sequencing since sequencing cannot distinguish infectious from non-infectious virions.

Mentions:
To confirm that the decreased MxA resistance of polymerase activity correlated with the effect of MxA on viral replication, we carried out competition experiments between wildtype and mutant viruses. Such competition experiments provide a sensitive and internally controlled way to measure the relative fitness of two viral variants. We used reverse genetics to generate influenza viruses carrying wildtype NP, the D51Dsyn mutation, or the D51N mutation (S7 Fig). We mixed each mutant virus with wildtype virus at a 1:1 ratio of infectious particles, and infected MxA-expressing or non-expressing cells at a low MOI. At 10 and 54 hours post-infection, we isolated viral RNA and determined the frequency of each variant by deep sequencing. As expected, the wildtype D51 variant greatly increased in frequency relative to the MxA-sensitive D51N mutant in MxA-expressing cells, whereas the two variants remained at similar frequencies in cells not expressing MxA (Fig 6). Also as expected, the wildtype D51 variant and its synonymous variant remained at roughly equal frequencies in the control competitions (Fig 6). This competition experiment verifies that an amino-acid mutation away from the wildtype identity at site 51 strongly increases MxA sensitivity as measured by viral growth.

ppat.1006288.g006: A mutation at site 51 in NP increases MxA sensitivity as measured by viral competition.We used reverse genetics to generate viruses carrying the wildtype NP, the D51N mutation, or a synonymous mutation at site 51. Each virus was generated in duplicate. We mixed the wildtype virus with one of the mutant viruses, infected MxA-expressing and non-expressing cells, and measured mutant frequencies after 10 and 54 hours. The plots show the frequency of the wildtype D51 variant relative to the N51 amino-acid mutant or the Dsyn51 synonymous mutant at each timepoint. In both replicates of the competition, the wildtype D51 variant was strongly favored over the N51 mutant in MxA-expressing cells. We targeted a 1:1 ratio of infectious particles in our initial inoculum, but this ratio was impossible to verify by direct sequencing since sequencing cannot distinguish infectious from non-infectious virions.

Mentions:
To confirm that the decreased MxA resistance of polymerase activity correlated with the effect of MxA on viral replication, we carried out competition experiments between wildtype and mutant viruses. Such competition experiments provide a sensitive and internally controlled way to measure the relative fitness of two viral variants. We used reverse genetics to generate influenza viruses carrying wildtype NP, the D51Dsyn mutation, or the D51N mutation (S7 Fig). We mixed each mutant virus with wildtype virus at a 1:1 ratio of infectious particles, and infected MxA-expressing or non-expressing cells at a low MOI. At 10 and 54 hours post-infection, we isolated viral RNA and determined the frequency of each variant by deep sequencing. As expected, the wildtype D51 variant greatly increased in frequency relative to the MxA-sensitive D51N mutant in MxA-expressing cells, whereas the two variants remained at similar frequencies in cells not expressing MxA (Fig 6). Also as expected, the wildtype D51 variant and its synonymous variant remained at roughly equal frequencies in the control competitions (Fig 6). This competition experiment verifies that an amino-acid mutation away from the wildtype identity at site 51 strongly increases MxA sensitivity as measured by viral growth.

The innate-immune restriction factor MxA inhibits influenza replication by targeting the viral nucleoprotein (NP). Human influenza virus is more resistant than avian influenza virus to inhibition by human MxA, and prior work has compared human and avian viral strains to identify amino-acid differences in NP that affect sensitivity to MxA. However, this strategy is limited to identifying sites in NP where mutations that affect MxA sensitivity have fixed during the small number of documented zoonotic transmissions of influenza to humans. Here we use an unbiased deep mutational scanning approach to quantify how all single amino-acid mutations to NP affect MxA sensitivity in the context of replication-competent virus. We both identify new sites in NP where mutations affect MxA resistance and re-identify mutations known to have increased MxA resistance during historical adaptations of influenza to humans. Most of the sites where mutations have the greatest effect are almost completely conserved across all influenza A viruses, and the amino acids at these sites confer relatively high resistance to MxA. These sites cluster in regions of NP that appear to be important for its recognition by MxA. Overall, our work systematically identifies the sites in influenza nucleoprotein where mutations affect sensitivity to MxA. We also demonstrate a powerful new strategy for identifying regions of viral proteins that affect inhibition by host factors.